Mono-ADP-ribosylation is a posttranslational modification of proteins in which ADP-ribose is transferred from NAD to protein substrates by amino acid specific ADP-ribosyltransferases. The modification can be reversed by ADP-ribosylarginine hydrolase which removes the ADP-ribose adduct from proteins. An arbinine-specific mono-ADP-ribosyltransferase was localized to the surface of differentiated mouse skeletal muscle cells, anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP- ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVMGAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha7. Following partial tryptic digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha7. Selective expression of both ADP-ribosyltransferase and integrin alpha7 in cardiac and skeletal muscle, similar developmental appearance and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.